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Artificial insemination is an important assisted reproductive technology that has been applied in several mammalian species. However, successful cryopreservation of semen of South American camelids has been limited, hindering the commercial application of artificial insemination in alpaca species. In this scenario, the addition of antioxidants to semen extenders provides a strategy to improve the freezability of mammalian sperm. Bioactive metabolites from natural extracts of black maca have shown valuable antioxidant properties. Thus, the objective of this study was to evaluate the effect of the addition of atomized black maca in the freezing medium of epididymal spermatozoa of alpacas. Fifteen pairs of epididymis were collected from a local slaughterhouse. Each sample was divided into six groups: (1) fresh, (2) yolk medium (YM), (3) 10 mg/mL maca, (4) 20 mg/mL maca, (5) 30 mg/mL maca, and (6) resveratrol (as an antioxidant control). Sperm cryopreservation was performed through the slow freezing method. Markers associated with functionality, such as motility, viability, and plasma membrane integrity, as well as markers associated with oxidative damage, such as DNA integrity, total ROS production, and mitochondrial function, were analyzed. The results show that the supplementation with black maca (20 mg/mL) improved the sperm motility, viability, plasma membrane integrity, and mitochondrial function evaluated according to an index of formazan deposits. Similarly, the ROS production decreased with maca at 20 mg/mL, although the DNA integrity did not show any differences among the groups. These results suggest that maca at 20 mg/mL has cytoprotective effects during freezing/thawing of epididymal sperm of alpaca species. Further research will be focused on assessing the effects of maca supplementation on semen extenders by using biomolecular markers (proAKAP4) associated with fertility.

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Male infertility is linked to several environmental and mutagenic factors. Most of these factors, i.e., lifestyle, radiations, and chemical contaminations, work on the fundamental principles of physics, chemistry, and biology. Principally, it may induce oxidative stress (OS) and produce free radicals within the cells. The negative effect of OS may enhance the reactive oxygen species (ROS) levels in male reproductive organs and impair basic functions in a couple's fertility. Evidence suggests that infertile men have significantly increased ROS levels and a reduced antioxidant capacity compared with fertile men. Although, basic spermatic function and fertilizing capacity depend on a delicate balance between physiological activity of ROS and antioxidants to protect from cellular oxidative injury in sperm, that is essential to achieve pregnancy. The ideal oxidation-reduction (REDOX) equilibrium requires a maintenance of a range of ROS concentrations and modulation of antioxidants. For this reason, the chapter focuses on the effects of ROS in sperm functions and the current concepts regarding the benefits of medical management in men with diminished fertility and amelioration of the effect to improve sperm function. Also, this evidence-based study suggests an increasing rate of infertility that poses a global challenge for human health, urging the need of health care professionals to offer a correct diagnosis, comprehension of the process, and an individualized management of the patients.

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STUDY QUESTION: Does oxidative stress (OS) activate autophagy in human sperm? SUMMARY ANSWER: Human spermatozoa subjected to OS activate an autophagic response. WHAT IS KNOWN ALREADY: Autophagy is a regulated pathway of lysosomal degradation which helps eukaryotic cells to maintain or restore homeostasis, being a cellular stress response mechanism. OS is a main cause of impaired sperm function and is linked to male infertility; however, whether OS activates autophagy in human spermatozoa is unknown. STUDY DESIGN, SIZE, DURATION: Human spermatozoa were exposed separately to ionomycin and hydrogen peroxide in order to induce OS. An untreated control group was included. Sperm cells under OS were then exposed to chloroquine in order to block autophagy. An untreated control and a control incubated only with the OS inducer were included in each experimental setting. PARTICIPANTS/MATERIALS, SETTING, METHODS: For this study, semen samples from normozoospermic donors were used and motile sperm cells were selected by the swim up technique. First, the generation of OS under our experimental conditions was demonstrated by analyzing sperm parameters including viability, reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) motility and thiol oxidation. Then, proteins involved in autophagy, including the microtubule-associated protein light chain 3 (LC3), particularly LC3-I and LC3-II, autophagy-related 5 (ATG5) and autophagy-related 16 (ATG16) proteins as well as the phosphorylated form of AMP-activated protein kinase (pAMPK) were evaluated in spermatozoa exposed to OS and compared to the untreated control. Finally, the impact of autophagy blocking by chloroquine treatment on sperm quality, metabolic parameters, including glycolysis and oxidative phosphorylation, as well as the cell death markers phosphatidylserine externalization and caspase activation was analyzed. Sperm quality parameters, cell death markers and autophagy-related proteins were analyzed by flow cytometry. Motility was evaluated by the computer-assisted sperm analysis system and metabolic parameters were analyzed using an extracellular flux analyzer. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to ionomycin and hydrogen peroxide promotes OS resulting in increased ROS production and decreased viability, ΔΨm and motility, while increasing thiol oxidation. These alterations were accompanied by a decrease in LC3-I, indicating that autophagy was activated upon OS exposure. Ionomycin also caused an increase in LC3-II, ATG5, ATG16 and pAMPK content. Autophagy blocking of sperm exposed to OS caused deterioration in sperm quality and metabolic parameters as well as an increase in cell death markers. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using motile sperm from normozoospermic donors; tests on sperm from infertile patients were not carried out. The autophagy blocking plus OS might generate a non-specific response to a highly stressful situation leading to the induction of cell death. WIDER IMPLICATIONS OF THE FINDINGS: Human spermatozoa subjected to OS activate an autophagic response and its blockage results in increased oxidative damage and commits spermatozoa to cell death. These results suggest a crucial role of autophagy as a stress response by male gametes, which contributes to maintaining the functionality and lifespan of ejaculated sperm cells. Detection of autophagy activation in sperm cells ex vivo could be included in semen analysis as a marker of OS, especially in men displaying high levels of seminal ROS. Novel strategies that aim to activate this cellular stress response could improve sperm quality/functionality under natural ejaculate conditions in which increased ROS levels are expected. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Fondo Nacional de Investigación Científica y Tecnológica, Chile (ANID/FONDECYT, Grant number 11170758 to P.U.); the Comisión Nacional de Investigación Científica y Tecnológica, Chile (ANID/CONICYT, Grant number PAI79160030 to P.U.) and the Dirección de Investigación, Universidad de La Frontera. The authors disclose no potential conflicts of interest.

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Kacang goats are small ruminants produced by low-income households in smallholder and farm to reduce poverty and prevent undernutrition. Studies to find a cryopreservation protocol for Kacang goat semen are expected to multiplication of genetically superior animals selected by the paternal lineage. This study evaluated the effect of thawing temperature and supplementation of the green tea extract nanoparticle in skim milk-egg yolk (SM-EY) extender on post-thaw sperm quality of Kacang goat semen. Six ejaculates of Kacang goat were diluted in SM-EY supplemented or not (control group) with 0.001 mg/mL NPs GTE. The diluted semen was packaged with 0.25 mL straws (insemination dose: 60x106 sptz/mL) and cryopreserved. Then, six samples of the control group and NPs GTE groups were thawed at 37°C or 39°C sterile water for 30 s and submitted to sperm quality evaluations. The sperm viability, motility, and intact of the plasma membrane (IPM) were higher (p<0.05) in NPs GTE group than control group. In contrast, the NPs GTE group presented lower (p<0.05) malondialdehyde levels and sperm DNA fragmentation (SDF) compared with the control group. The catalase levels were not significantly different (p > 0.05) between the control and NPs GTE groups. Thawing at 39°C resulted in higher (p<0.05) sperm viability, motility, and IPM than thawing at 37°C. However, thawing at 39°C group presented lower (p<0.05) malondialdehyde levels compared with thawing at 37°C. SDF and catalase levels were similar (p>0.05) between thawing at 37°C and thawing at 37°C. In conclusion, supplementation of 0.001 mg/mL of NPs GTE in SM-EY extender and thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.

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OBJECTIVE: Approximately 15% of the couples suffer from infertility. Half of the cases of infertility are due to male factors. Several sperm function tests have been proposed to evaluate male fertility, but sperm analysis is still the first and most important diagnostic test for male infertility. The prognostic value of semen characteristics such as concentration, morphology and motility markers are often confused with male infertility. Evaluation of seminal parameters and classification for normality remains a frequent topic of discussion. METHODS: This study evaluated 477 semen samples from men undergoing investigation or infertility treatment between 2011 and 2015. RESULTS: The spermograms of 401 patients were deemed abnormal based on the 1999 World Health Organization (WHO) criteria; the number changed to 223 when the spermograms were assessed based on the 2010 WHO criteria and to 200 when Total Motile Sperm Count (TMSC) was used as the criterion. Sperm morphology was the item in the criteria that most significantly changed spermogram classification. Normality parameters became less rigid from 1999 to 2010, thereby significantly changing the proportion of individuals no longer described as infertile/subfertile. CONCLUSIONS: The classification based on TMSC could not differentiate between fertile and infertile subjects for not taking sperm morphology into account. Nevertheless, it may be helpful in cases where intrauterine insemination is indicated.

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Kacang goats are small ruminants produced by low-income households in smallholder and farm to reduce poverty and prevent undernutrition. Studies to find a cryopreservation protocol for Kacang goat semen are expected to multiplication of genetically superior animals selected by the paternal lineage. This study evaluated the effect of thawing temperature and supplementation of the green tea extract nanoparticle in skim milk-egg yolk (SM-EY) extender on post-thaw sperm quality of Kacang goat semen. Six ejaculates of Kacang goat were diluted in SM-EY supplemented or not (control group) with 0.001 mg/mL NPs GTE. The diluted semen was packaged with 0.25 mL straws (insemination dose: 60x106 sptz/mL) and cryopreserved. Then, six samples of the control group and NPs GTE groups were thawed at 37°C or 39°C sterile water for 30 s and submitted to sperm quality evaluations. The sperm viability, motility, and intact of the plasma membrane (IPM) were higher (p<0.05) in NPs GTE group than control group. In contrast, the NPs GTE group presented lower (p<0.05) malondialdehyde levels and sperm DNA fragmentation (SDF) compared with the control group. The catalase levels were not significantly different (p > 0.05) between the control and NPs GTE groups. Thawing at 39°C resulted in higher (p<0.05) sperm viability, motility, and IPM than thawing at 37°C. However, thawing at 39°C group presented lower (p<0.05) malondialdehyde levels compared with thawing at 37°C. SDF and catalase levels were similar (p>0.05) between thawing at 37°C and thawing at 37°C. In conclusion, supplementation of 0.001 mg/mL of NPs GTE in SM-EY extender and thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.(AU)

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The giant anteater (Myrmecophaga tridactyla) is being threatened by natural habitat destruction and fragmentation, illegal hunting and road kills. In this context, the generation of basic information on the reproductive parameters of this species is vital, aiming to improve reproductive management via, amongst others, assisted reproductive technologies. This study aimed to describe the morphological and functional features of semen collected from captive giant anteaters. Electroejaculation was performed in 13 animals housed in zoos located in São Paulo state, Brazil. Semen samples were collected from 13 animals in 16 procedures. Samples were evaluated for volume, motility, vigor, pH, concentration, sperm morphology, and functional tests. The following mean values were obtained: volume 1.28 ± 0.27 mL; motility 28.3 ± 6.2%; vigor 2.4 ± 0.25; concentration 129.4 ± 36.1 × 106 sperm/mL; pH 7.4 ± 0.2. Total acrosome, head, midpiece, and tail sperm abnormalities were 3.2 ± 0.8%, 25.4 ± 3.6%, 20.7 ± 3.2%, and 14.7 ± 2.6%, respectively. Intact acrosome was found in 83.7 ± 3.1% and intact membrane in 81.1 ± 4.0% of all samples collected. Mitochondrial activity was 66.4 ± 6.0% (Class I), 18.7 ± 2.9% (Class II), 8.0 ± 2.0% (Class III), 3.9 ± 1.0% (Class IV), and 3.0 ± 0.9% (Class V). Sperm DNA fragmentation rate was 13.2 ± 3.7%. These results indicated that electroejaculation is a feasible method for semen collection in giant anteaters, allowing a more detailed description of the semen in this species.

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Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction. In this review we will focus on the assays routinely performed in our laboratories, giving a literature support to critically analyse different approaches. In fact, researchers usually tend to look for the "one shot" parameter that could explain itself a specific process; it is our conviction that a multiparametric approach is still more valid, as some changes in sperm function are very complex and could be explained only by operating in different ways. Sperm motility, the most evident sperm characteristic, should be assessed by computer-aided sperm analysers that permit an objective evaluation of the motility and its kinematic parameters. Commercial and open source instruments are available and could be profitably used together with specific statistical approaches. The use of microscopy, and particularly fluorescent microscopy, could be a very useful tool to assess different parameters in sperm cells both by fluorophores that give indication of a determined function, and by immunolocalization of proteins, that permits the discover of new features or to explain particular sperm functions. The same substrates could be used also in flow cytometry: the difference is that it permits to study wider sperm populations (and their sub-population distribution). Flow cytometry is undergoing a very wide use in spermatology and technical and experimental rigor is needed to obtain reliable results. Metabolic assessment of sperm features, particularly energetic supply, ATP formation and other enzyme activities, could represent a very important challenge to acquire new information and complete/integrate those derived from other techniques. Finally, functional assays such as oocyte binding and in vitro fertilization, represent a very strong tool to assess sperm function in vitro, as they could evidence the functional intactness of some pathways.

RESUMO

In vitro manipulation of spermatozoa leads to deleterious changes of structure and function that occur mainly due to oxidative stress, therefore, prevention or treatment is a strategy to improve the functions of processed sperm. In the present study, the aim was to evaluate the effects of MnTBAP supplementation, a compound with antioxidant activity, on in vitro capacitation conditions of thawed equine sperm. For this purpose, stallion spermatozoa (2 × 106 cells/mL) were incubated in the sperm-TLP base medium for 4 h in which there were three different conditions: non-capacitating, capacitating, and capacitating plus 150 mM MnTBAP. There were incubations for 4 h at 37.5 °C in a humidified air atmosphere. Sample analysis was performed immediately after thawing (0 h), and at the end of the incubation period (4 h), unless otherwise indicated. The following variables were evaluated for spermatozoa: plasma membrane integrity and fluidity, acrosome integrity, intracellular calcium concentrations, intracellular pH, tyrosine phosphorylation, ATP concentrations, motility and heterologous zona-binding assay, using flow cytometry, fluorescent microscopy and/or chemiluminescence, depending on the most appropriate procedure for the variable being evaluated. Results indicated that capacitation-like changes were synergistically induced by the cAMP agonists, phosphodiesterase inhibitor and bicarbonate. The presence of bovine serum albumin was harmful to the plasma membrane. The MnTBAP supplementation had a positive effect on viability-related markers (plasma membrane integrity, membrane fluidity, associated with greater intracellular pH) when there were capacitating conditions. In conclusion, the activity of MnTBAP contributes to improving the in vitro incubation conditions of frozen-thawed stallion sperm.

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Male infertility is an increasing health problem, and oxidative/nitrosative stress plays an important role in the etiology of this condition. Nitrosative stress due to excessive levels of reactive nitrogen species (RNS) is associated with impaired male fertility. Flow cytometry may be a useful tool for semen evaluation, but the availability of multiparameter assays for analysis of sperm quality is limited. The present study standardized a multiparameter flow cytometry analysis for nitrosative stress status in human spermatozoa in a single assay. A suitable multicolor fluorochrome panel was designed and consisted of fluorescein-boronate to detect peroxynitrite, a highly RNS, propidium iodide to analyze viability, tetramethylrhodamine methyl ester perchlorate to detect mitochondrial membrane potential (MMP) and monobromobimane to analyze thiol oxidation. Proper positive and negative controls for each fluorochrome were used to establish the technique, and sperm cells of different qualities and spermatozoa subjected to cryopreservation were analyzed. The results showed that the controls clearly discriminated between the high and low fluorescence intensities for each fluorochrome. The analysis of sperm cells of different quality demonstrated that the assay properly detected differences in all parameters analyzed according to sperm quality. The results may be reported as the mean fluorescence intensity of each fluorochrome and the percentage of cells exhibiting different characteristics. In conclusion, a protocol was standardized to analyze nitrosative stress status, including peroxynitrite production, viability, MMP, and thiol oxidation, in a single analysis using flow cytometry. This protocol may be applied to research approaches and clinical andrology to improve the evaluation of sperm quality and provide a promising tool to increase the use of clinical flow cytometry. © 2020 International Society for Advancement of Cytometry.

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Oxidative stress contributes importantly to the aetiology of male infertility, impairing sperm function. The protective effect of antioxidants on seminal parameters has been established, and the antioxidant penicillamine has shown beneficial effects; however, its protective effect on human spermatozoa exposed to oxidative stress has not been reported. The objective of this work was to evaluate the effect of penicillamine on human spermatozoa exposed in vitro to oxidative stress. First, the effect of penicillamine on spermatozoa from normozoospermic donors was evaluated. Then, the effect of penicillamine on spermatozoa exposed to oxidative stress induced separately by ionomycin and hydrogen peroxide (H2 O2 ) was analysed. An untreated control and a control treated only with the oxidative stress inducer were included. Reactive oxygen species (ROS) levels, viability, mitochondrial membrane potential (MMP) and motility were analysed. The results showed that penicillamine, added to the incubation medium, decreased the ROS levels induced by ionomycin and H2 O2 , and this effect was associated with better preservation of MMP, motility, and ATP levels. These results highlight the potential advantages of penicillamine supplementation of sperm culture medium, especially for semen samples with high ROS levels and also in circumstances where laboratory handling can cause an increase in ROS production.

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Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) that is widely used in the manufacturing of plastics and inner linings of food cans. Previously, it was reported that BPA disturbed the sexual dimorphic nucleus of the hypothalamus and delaying the onset of puberty attributed to an estrogenic action. In addition, BPA during the perinatal period increased LH serum concentrations in male offspring of dams at doses below the reproductive NOAEL (No Observable Adverse Effect Level) based upon World Health Organization guidelines. Based upon these findings, the objective of this study was to (1) determine the effects of perinatal treatment with low doses of BPA on regulation of spermatogenesis in adult offspring and (2) elucidate molecular mechanisms involved in the pathogenesis of gonadal dysfunction. The expression of genes related to spermatogenesis was disrupted with adverse consequences on sperm production, reserves, and function. Both BPA treated groups exhibited reduction in sperm production and epithelial height of seminiferous tubules, accompanied by diminished integrity of the acrosome and plasma membrane, decreased mitochondrial activity and increased incidence of morphological abnormalities. The sperm transit time was also slower. However, only in the group receiving the higher BPA dose was transcript expression of genes affected (reduced Ar and increased Esr1). It is of interest that serum testosterone levels were elevated in the same group where Ar was decreased. Data suggest that exposure to low BPA doses during hypothalamic sexual differentiation period produces permanent deleterious effects on spermatogenesis in adulthood.

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Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction. In this review we will focus on the assays routinely performed in our laboratories, giving a literature support to critically analyse different approaches. In fact, researchers usually tend to look for the “one shot” parameter that could explain itself a specific process; it is our conviction that a multiparametric approach is still more valid, as some changes in sperm function are very complex and could be explained only by operating in different ways. Sperm motility, the most evident sperm characteristic, should be assessed by computer-aided sperm analysers that permit an objective evaluation of the motility and its kinematic parameters. Commercial and open source instruments are available and could be profitably used together with specific statistical approaches. The use of microscopy, and particularly fluorescent microscopy, could be a very useful tool to assess different parameters in sperm cells both by fluorophores that give indication of a determined function, and by immunolocalization of proteins, that permits the discover of new features or to explain particular sperm functions. The same substrates could be used also in flow cytometry: the difference is that it permits to study wider sperm populations (and their subpopulation distribution). Flow cytometry is undergoing a very wide use in spermatology and technical and experimental rigor is needed to obtain reliable results. Metabolic assessment of sperm features, particularly energetic supply, ATP formation and other enzyme activities, could represent a very important challenge to acquire new information and complete/integrate those derived from other techniques. Finally, functional assays such as oocyte binding and in vitro fertilization, represent a very strong tool to assess sperm function in vitro, as they could evidence the functional intactness of some pathways.

Assuntos

RESUMO

Different approaches can be used to assess sperm function in different conditions, i.e. sperm storage, freezing-thawing or activation by induction of capacitation and acrosome reaction. In this review we will focus on the assays routinely performed in our laboratories, giving a literature support to critically analyse different approaches. In fact, researchers usually tend to look for the “one shot” parameter that could explain itself a specific process; it is our conviction that a multiparametric approach is still more valid, as some changes in sperm function are very complex and could be explained only by operating in different ways. Sperm motility, the most evident sperm characteristic, should be assessed by computer-aided sperm analysers that permit an objective evaluation of the motility and its kinematic parameters. Commercial and open source instruments are available and could be profitably used together with specific statistical approaches. The use of microscopy, and particularly fluorescent microscopy, could be a very useful tool to assess different parameters in sperm cells both by fluorophores that give indication of a determined function, and by immunolocalization of proteins, that permits the discover of new features or to explain particular sperm functions. The same substrates could be used also in flow cytometry: the difference is that it permits to study wider sperm populations (and their subpopulation distribution). Flow cytometry is undergoing a very wide use in spermatology and technical and experimental rigor is needed to obtain reliable results. Metabolic assessment of sperm features, particularly energetic supply, ATP formation and other enzyme activities, could represent a very important challenge to acquire new information and complete/integrate those derived from other techniques. Finally, functional assays such as oocyte binding and in vitro fertilization, represent a very strong tool to assess sperm function in vitro, as they could evidence the functional intactness of some pathways.(AU)

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Low levels of reactive oxygen species (ROS) in sperm are essential for various sperm functions such as capacitation, hyperactivation and acrosome reaction. However, increased synthesis of ROS or a disruption of antioxidative status (e.g. in cryopreserved sperm) can induce oxidative stress (OS). Sperm are particularly vulnerable to OS, as their plasma membrane contains large amounts of polyunsaturated fatty acids and they have limited antioxidative capacity (due to low cytoplasmic volume). Oxidative stress disturbs sperm function by damaging sperm proteins, lipids and DNA. Under relatively low OS sperm may retain their fertilizing ability, which might result in transfer of impaired paternal molecules (e.g. damaged DNA) to the fertilized oozyte. Oocytes can repair damaged paternal DNA, but only to a certain extent. Most embryos are either repaired (based on limited DNA damage in blastocysts) or eliminated (based on low percentage of blastocyst formation when sperm with damaged DNA is used for fertilization). However, some blastocysts had increases in both DNA damage and apoptosis, which could have important implications for subsequent development. In several studies, exogenous antioxidants improved quality of sperm exposed to oxidative stress and subsequent embryo development. However, there is still a knowledge gap regarding whether these alterations affect embryonic survival and further development to a live fetus and healthy offspring.

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The knowledge of sperm quality in the broodstock males of different ages is a prerequisite to identify the reproductive ability of cultivated fish for the hatchery management. Thus, in this work, we analysed sperm function of the semen stored of broodstock males of rainbow trout (Oncorhychus mykiss) in different reproductive ages (2, 3 and 4 years old). Sperm samples of each reproductive age were stored in Storfish® during 10 days at 4°C, and then, motility, viability, mitochondrial function (MMP), superoxide anion (O2-) level and DNA fragmentation (DNAfrag ) were assessed. The results demonstrated that sperm function parameters were affected significantly by the age of the males and the time of storage. Motility, viability and MMP significantly decreased, and DNAfrag and O2- level increased with the age increment and the time of storage. In conclusion, sperm quality of 2 and 3 years old were superior to those of 4 years old, based on higher quality of various sperm functions such as motility, viability, MMP, DNA integrity and level O2- during short-term storage. This information must be considered for optimum utilization of broodstock males in aquaculture.

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RESUMO

Low levels of reactive oxygen species (ROS) in sperm are essential for various sperm functions such as capacitation, hyperactivation and acrosome reaction. However, increased synthesis of ROS or a disruption of antioxidative status (e.g. in cryopreserved sperm) can induce oxidative stress (OS). Sperm are particularly vulnerable to OS, as their plasma membrane contains large amounts of polyunsaturated fatty acids and they have limited antioxidative capacity (due to low cytoplasmic volume). Oxidative stress disturbs sperm function by damaging sperm proteins, lipids and DNA. Under relatively low OS sperm may retain their fertilizing ability, which might result in transfer of impaired paternal molecules (e.g. damaged DNA) to the fertilized oozyte. Oocytes can repair damaged paternal DNA, but only to a certain extent. Most embryos are either repaired (based on limited DNA damage in blastocysts) or eliminated (based on low percentage of blastocyst formation when sperm with damaged DNA is used for fertilization). However, some blastocysts had increases in both DNA damage and apoptosis, which could have important implications for subsequent development. In several studies, exogenous antioxidants improved quality of sperm exposed to oxidative stress and subsequent embryo development. However, there is still a knowledge gap regarding whether these alterations affect embryonic survival and further development to a live fetus and healthy offspring.

Assuntos

RESUMO

Low levels of reactive oxygen species (ROS) in sperm are essential for various sperm functions such as capacitation, hyperactivation and acrosome reaction. However, increased synthesis of ROS or a disruption of antioxidative status (e.g. in cryopreserved sperm) can induce oxidative stress (OS). Sperm are particularly vulnerable to OS, as their plasma membrane contains large amounts of polyunsaturated fatty acids and they have limited antioxidative capacity (due to low cytoplasmic volume). Oxidative stress disturbs sperm function by damaging sperm proteins, lipids and DNA. Under relatively low OS sperm may retain their fertilizing ability, which might result in transfer of impaired paternal molecules (e.g. damaged DNA) to the fertilized oozyte. Oocytes can repair damaged paternal DNA, but only to a certain extent. Most embryos are either repaired (based on limited DNA damage in blastocysts) or eliminated (based on low percentage of blastocyst formation when sperm with damaged DNA is used for fertilization). However, some blastocysts had increases in both DNA damage and apoptosis, which could have important implications for subsequent development. In several studies, exogenous antioxidants improved quality of sperm exposed to oxidative stress and subsequent embryo development. However, there is still a knowledge gap regarding whether these alterations affect embryonic survival and further development to a live fetus and healthy offspring.(AU)

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Long term storage of canine frozen semen is conventionally performed in liquid nitrogen (LN2). However, previous works in freezing canine semen using a -80 °C ultra-freezer (-80°C-UF) showed no differences on sperm quality after thawing. The main objective of this study was to compare the effects of the freezing techniques using LN2 or -80°C-UF on sperm function and in vivo fertility of frozen-thawed dog semen. The sperm-rich fraction of the ejaculate was collected separately from five Chihuahua breed, and each one divided into two aliquots, and frozen and stored in LN2 or -80°C-UF. Sperm function was analyzed for motility and viability, acrosome integrity, mitochondrial function and phosphatidylserine translocation by flow cytometry before and after cryopreservation. A total of 10 bitches were intravaginal inseminated (IVAI; LN2 frozen-thawed semen = 5 and -80°C-UF frozen-thawed semen = 5). Pregnancy status was confirmed 30 d after IVAI by transabdominal ultrasonography and live born puppies at term were recorded. Sperm function parameters were affected for both freezing protocols. Differences (P < 0.05) were found between freezing and storage methods in most of the parameters of sperm function analyzed, except in the phosphatidylserine translocation. The percentages of pregnancies were not different between the two freezing and storage protocols used. Semen freezing and storage using -80 °C UF is an effective technique for long-term preservation of canine spermatozoa.

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The most toxic species in live systems include reactive nitrogen species such as peroxynitrite, which at high levels induces nitrosative stress. In human spermatozoa, the negative effect of peroxynitrite on motility and mitochondrial membrane potential was recently demonstrated, and the hypothesis of this work is that impairment of ATP production could be one cause of the effect on motility. Therefore, the aim here was to evaluate ATP production by both glycolysis and oxidative phosphorylation (OXPHOS) in spermatozoa exposed to peroxynitrite in vitro. Human spermatozoa were incubated with SIN-1, a molecule which generates peroxynitrite, and the ATP level was evaluated. Then, to inactivate glycolysis or OXPHOS, spermatozoa were incubated with pharmacological inhibitors of these pathways. Spermatozoa treated for inactivating one or the other pathway were exposed to SIN-1, and the ATP level was compared to the control without SIN-1 in each condition. The ATP level fell after peroxynitrite exposure. The ATP in spermatozoa treated for inactivating one or the other metabolic pathway and subsequently exposed to peroxynitrite was reduced compared with the control. These results show for the first time that an important mechanism by which peroxynitrite reduces sperm function is the inhibition of ATP production, affecting both glycolysis and OXPHOS.

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